Trichomonas tenax induces barrier defects and modulates the inflammatory cytotoxicity of gingival and pulmonary epithelial cells

Background: Trichomonas tenax is a single-cell flagellated anaerobic organism, commensal in the human oral cavity. Although a previous study indicated that T. tenax could cause cell damage and phagocytose host epithelial cells, its pathological effects on gum cells remain unknown. Furthermore, several case reports have detected T. tenax in several patients with empyema and/or pleural effusion, which may have been aspirated from the oral cavity. However, the cytotoxic effects and immune responses of alveolar cells are unknown. Therefore, we aimed to determine the cytotoxic and immune effects of T. tenax on gums and pulmonary cell lines. The cytopathic effect and lactate dehydrogenase (LDH) cytotoxicity assays were used to determine the level of cell damage in gum and lung epithelial cells. Western blot was used to determine the disruption of cell junctions. Finally, epithelial cell cytokines were measured using ELISA to elucidate the immune response to T. tenax. Results: We found that T. tenax induced a cytotoxic effect on gum epithelial cells by disrupting cell junctions; however, it hardly triggered cellular damage in alveolar A549 cells and mucoepidermoid NCI-H292 cells. Furthermore, T. tenax induced the production of IL-6 at a low multiplicity of infection (MOI) in gum, A549, and NCI-H292 cells. Conclusions: Our results suggest that T. tenax can trigger gingival cell cytotoxicity, disrupt cell junctions, and induce IL-6 production in gingival and pulmonary cell lines.


Introduction
Trichomonas tenax is a single-cell flagellated anaerobic organism. It is typically found in the human oral cavity and is transmitted via saliva droplets, drinking water, and contaminated food [14]. Trichomonas tenax is a known oral commensal protozoa and is distributed widely among 4-53% of the population [11]. However, studies have indicated that the detection rate is higher in patients with poor oral hygiene, and its presence was highly correlated with periodontal disease in a cohort study [2,11]. Based on a systematic review and meta-analysis, T. tenax was shown to have a relationship with candidiasis, gingivitis and periodontitis in pooled prevalence [8]. In addition, a previous study showed a relationship between the severity of periodontitis and the detection of T. tenax in dental plaque [16]. However, the pathogenicity and virulence of T. tenax are still unknown. Therefore, it is crucial to determine the interaction between T. tenax and gum tissue.
In a previous study, transmission electron microscopy (TEM) revealed that T. tenax could attach to mammalian cells and form aggregates that coated the surface of epithelial cells until disruption of the monolayer [18]. Furthermore, cytotoxicity and phagocytosis were observed in Madin-Darby canine kidney (MDCK) and HeLa cells during incubation with T. tenax. Similar results were observed in the presence of T. vaginalis and host cell incubation [18]. Although T. tenax induces cell damage, the exact oral pathology underlying this interaction between T. tenax and gum cells remains unclear. Previous studies have shown that most cases of bacterial pneumonia result from oral and/or pharyngeal flora that are obtained primarily by aspiration and inhalation, especially in patients with periodontal disease or poor oral hygiene [15,19,20]. Therefore, the presence of oral pathogens could increase the risk of pneumonia in elderly or immunodeficient patients following aspiration or inhalation. Trichomonas tenax is the most frequent species of trichomonads that causes pulmonary trichomoniasis accompanied by pyopneumothorax and empyema [26,27]. Sequencing analysis showed that almost 30% of bronchoalveolar lavage fluid samples from 77 patients in the ICU contained T. tenax. Additionally, among these confirmed cases, 17 patients were also associated with acute respiratory distress syndrome (ARDS) [6]. Although many clinical cases of T. tenax in the respiratory tract are associated with aspiration pneumonia, the process of pulmonary immunity and pathology during T. tenax invasion remains largely unknown. A previous study showed that incubation with the lysate of T. tenax directly stimulated the production of interleukin-8 in THP-1 cells [9]. However, the link between T. tenax and lung epithelial cells remains unknown. Therefore, it is necessary to determine the interaction between T. tenax and pulmonary epithelial cells to identify the role of T. tenax in pulmonary immunity and pathology.
In this study, we analyzed the integration of the epithelial barrier and polarity following the incubation of human gingival epithelial Smulow-Glickman (S-G) cells with T. tenax. Our results revealed that host cells lost tight junctions and showed reduced viability during coculture with T. tenax, as shown by western blot and cytopathic assays. Additionally, we demonstrated the production of proinflammatory cytokines by pulmonary epithelial cells after coculture with T. tenax. Our findings indicated that IL-6 was induced in the absence of cell damage, which indicates a role of lung immunity in pulmonary trichomoniasis.

Host cells and Trichomonas tenax
Human gingival epithelial Smulow-Glickman (S-G) cells (provided by Dr. Jenn-Wei Chen, Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan), A549 lung cancer cells (ATCC_CCL-185), and NCI-H292 mucoepidermoid pulmonary cells (ATCC_ CRL-1848) were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco TM , Thermo Fisher Scientific, Waltham, MA, USA) or Roswell Park Memorial Institute (RPMI 1640 Medium, Gibco TM , Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco TM , Thermo Fisher Scientific) and 100 units/mL penicillin-streptomycin (Thermo Fisher Scientific) and were maintained in a humidified atmosphere with 5.0% CO 2 at 37°C. Trichomonas tenax (ATCC_30207) was cultured in 20 mL of YI-S medium (yeast extract, iron-serum) supplemented with 10% heat-inactivated horse serum (Gibco TM , Thermo Fisher Scientific) in culture flasks under anaerobic conditions at 37°C and was harvested in the logarithmic phase after incubation for 24 h.
Host cell-T. tenax coculture conditions S-G, A549, and NCI-H292 cells were harvested and seeded in 6-or 24-well plates overnight in 5.0% CO 2 at 37°C to form a monolayer. For coculture conditions, T. tenax was harvested from the culture medium. Next, host cells and T. tenax at MOIs of 1, 2, 4, or 8 were cocultured in DMEM or RPMI medium without FBS and antibiotics for 24 h. The coculture medium was collected for immunoassays or cell-mediated cytotoxicity assays, and the remaining cells were used for cytopathic effect assays or observed with a Cell R microscope (Olympus CellR, Tokyo, Japan).

Cytopathic effect (CPE) assay
After host cells and T. tenax were cocultured for 24 h, the supernatant was removed, and the remaining cells were washed gently with 1 mL of phosphate-buffered saline (PBS). The cells were fixed with methanol and acetic acid at a ratio of 3:1 for 30 min. After air drying, Giemsa buffer (Merck, Darmstadt, Germany) was mixed with PBS buffer at a ratio of 9:1, added to 200 lL and incubated for 30 min. The wells were then rinsed with ddH 2 O and allowed to air dry. The results were observed using a Cell R microscope (Olympus CellR, Tokyo, Japan) and quantified using ImageJ software.

Lactate dehydrogenase (LDH) cytotoxicity assay
The coculture medium was examined by a CytoTox 96 Ò Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA). The medium was centrifuged at 250 Âg for 4 min to obtain a supernatant that was mixed with the CytoTox 96 Reagent. The mixture was protected from light for 30 min until the addition of the stop solution. The absorbance was recorded at 490 nm using a microplate spectrophotometer (Thermo Fisher Scientific) and analyzed using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA).

Enzyme-linked immunosorbent assay (ELISA)
The coculture supernatants were used to measure human IL-6, IL-1b, and TNFa levels using ELISA kits (BioLegend, San Diego, CA, USA). The samples were centrifuged at 250 Âg for 4 min, and the supernatant was tested by ELISA, according to the manufacturer's instructions. The absorbance was recorded at 450 nm and 570 nm using a microplate spectrophotometer (Thermo Fisher Scientific) and graphed using GraphPad Prism 7 software.

Statistical analysis
The data are presented as the mean ± standard deviation (SD). All comparisons were conducted using an unpaired two-tailed Student's t test. Statistical significance was set at p < 0.05. The data were analyzed statistically using GraphPad software (version 7.0).

Results
Trichomonas tenax could disrupt the monolayers of MDCK and HeLa cells and induce host cell damage. Therefore, in this study, we investigated the viability of S-G cells during incubation with T. tenax. The coculture conditions were 6, 12, and 24 h and MOIs of 1, 2, 4, and 8. The cytotoxicity of S-G cells gradually increased by incubation with T. tenax at MOIs of 4 and 8 for 12 to 24 h (Fig. 1A). As indicated by the white arrow in Figure 1A, shrinkage and rounding of S-G cells were observed at MOI 4 and MOI 8 after incubation for 24 h and 12 h, respectively. It was further observed that most of the cells detached from the bottom of the well after incubation with an MOI of 8 for 24 h (Fig. 1A). Furthermore, approximately 50% CPE was observed at an MOI of 4 and 8 in S-G cells after 24 h of incubation by staining with Giemsa buffer (Figs. 1B and 1C). Trichomonas tenax further induced the gradual release of LDH from S-G cells at MOIs of 2-8, which is consistent with the CPE results (Fig. 1D). In addition, T. tenax had little effect on LDH release in this coculture system. Therefore, the results of the present study indicate that T. tenax at MOIs of 4 and 8 triggers cell damage in S-G cells.
Due to the cytopathic effect of T. tenax, we examined whether cell junctions, which play important roles in the regulation of oral barrier function, were destroyed by T. tenax. The integration of cell junctions such as tight junctions, adherens junctions, desmosomes, and gap junctions was analyzed. The western blot results showed that T. tenax at an MOI of 4 dramatically decreased the expression of ZO-1, JAM-A, E-cadherin, b-catenin, desmoplakin 1 and 2, and connexin-43 in S-G cells (Fig. 2). In addition to the destruction of the cell barrier, the release of cytokines is an important factor in the onset of oral infection for disease progression. Our data showed that IL-6 was significantly higher in the MOI 1 and 2 groups than in the mock group after S-G cells were cocultured with T. tenax for 24 h (Fig. 3E). However, IL-1b was hardly detected in any of the groups after the incubation of S-G cells with T. tenax (Fig. 3F). TNFa, which was not shown in our data, was also undetectable by ELISA, regardless of the MOI. Taken together, these results indicate that T. tenax could induce the release of IL-6 from S-G cells, whereas IL-1b and TNFa were hardly induced in gum epithelial cells. These data reveal that T. tenax not only induces cytopathology but also promotes the release of proinflammatory cytokines from gum epithelial cells in the human oral cavity.
A previous clinical case report showed that patients who had pulmonary trichomoniasis exhibited sepsis-like syndrome and bronchospasm. In addition, some patients with late ARDS and empyema were shown to have T. tenax in their bronchoalveolar lavage. In addition to the damage induced by T. tenax in S-G cells, we further evaluated the cytopathic effect and cytokine production in A549 and NCI-H292 epithelial cells in the presence of T. tenax. There was no significant difference in the cytopathic effect on either cell line in response to MOIs of 1, 2, 4, and 8 (Figs. 3A, 3B, 3D). In addition, cell junctions were tightly bound under all conditions. Next, we investigated whether T. tenax induced cytokine production in A549 and NCI-H292 cells. Our data showed that IL-6 production was  induced in both cell types after T. tenax stimulation for 24 h (Figs. 3G, 3I). However, IL-1b was barely induced in any of the groups of A549 and NCI-H292 cells after incubation with T. tenax (Figs. 3H, 3J). TNFa was below the detection value in A549 and NCI-H292 cells. These data suggest that T. tenax has little effect on cytopathology, but can induce IL-6 secretion in pulmonary cell lines. This finding indicates that proinflammatory cytokines are released and induce downstream immunity following the invasion of T. tenax through the pulmonary barrier.

Discussion
In this study, we investigated the pathological and immunological effects of T. tenax on gum S-G cells and alveolar A549 cells. Our results showed that T. tenax triggered cytotoxic effects on S-G cells with increasing MOI levels. We further found that cell junctions, including tight junctions, adherens junctions, desmosomes, and gap junctions, were compromised when epithelial barriers were damaged (Fig. 4). These data correspond with the cytopathic effect of T. tenax on MDCK and HeLa cells reported in a previous study [18]. In addition, the data suggested that T. tenax could induce the immune response in the epithelium and was associated with periodontitis. Although several studies have previously reported the symptoms of pulmonary trichomoniasis, the underlying pathological mechanism remains unknown [11,13,14]. Our research showed that T. tenax did not damage A549 and NCI-H292 cells and did not induce the disruption of cell junctions (Fig. 4). However, cytokines were produced after incubation with T. tenax, which may be linked to the related immune response involving neutrophil recruitment and empyema in the pulmonary system [12,14].
Several studies have shown that several proteases isolated from clinical samples of T. tenax were identified by protein profiling [7,21]. These proteases were also shown to have proteolytic activity, which may promote their pathogenicity in oral and pulmonary invasion [7,21]. Cysteine proteinases (CPs) which are the most abundant soluble secretory proteins of Trichomonas vaginalis play important roles in cytotoxicity, adhesion, and apoptosis [17,24]. Monolayer HeLa cells were treated with CP65 of T. vaginalis, and cellular pathology was induced [1]. Furthermore, it was shown that CP30 of T. vaginalis could trigger apoptosis in human vaginal epithelial cells (HVECs) [22]. Another study indicated that incubation with T. vaginalis resulted in the disruption of junctional complex proteins in Caco-2 cells, while the same effect was observed in IPEC-J2 cells incubated with T. foetus [4,25]. Therefore, the function and activity of the cysteine proteinases of T. tenax need to be verified.
When challenged with Trichomonas vaginalis, the production of IL-6 in RWPE-1 cells was increased [10]. These results are similar to our data showing that IL-6 was produced by S-G, A549, and NCI-H292 cells that were incubated with T. tenax. A previous study showed that several oral pathogenic bacteria could induce the release of inflammatory cytokines from primary human gingival epithelial cells [23]. Interestingly, one study revealed that Aggregatibacter actinomycetemcomitans, which is associated with periodontal inflammation, could induce a low IL-1b response and high IL-6 response [23]. Therefore, our results might indicate a unique cytokine hallmark during the incubation of gum cells with T. tenax. Regarding the pulmonary system, several case reports have shown that patients infected with T. tenax had empyema and leukocytosis [12]. Furthermore, it has been reported that pleural effusion and high leukocyte counts were highly correlated with IL-6 concentrations [5]. In allergies, the cysteine protease produced by house dust mites (HDM), Der p 1, can cleave several cellular junctions to penetrate the subepithelial tissue and induce an immune response [3]. Similar to the results of our study, Der p 1 can trigger the production of IL-6 in the airway epithelium [3]. Several studies have also revealed that lysates from clinically isolated T. tenax exert proteolytic activity and contain several cysteine proteases ranging in size from 14 to 66 kDa [7,21]. These proteases may play roles in the induction of cell damage and cytokine production in these epithelial cells. Thus, more detailed experiments are required to determine the correlation between pulmonary immunology and T. tenax infection.

Conclusions
Our data have shown that T. tenax can induce a cytopathic effect on gingival cells. In addition, several cell-cell junctions were disrupted by an MOI of 4. In contrast, pulmonary epithelial cells were hardly damaged after incubation. Proinflammatory cytokines, especially IL-6, were released from gum and pulmonary epithelial cells after coculture with T. tenax. Therefore, our data indicate that T. tenax damages different cells to different degrees, but all contribute to the induction of a similar inflammatory response.